Oncolytic virus

ABSTRACT

The present invention is directed to a method of reducing the viability of a tumor cell involving administering a virus that is not a common human pathogen to the tumor cell. Preferably, the virus exhibits differential susceptibility, in that normal cells are not affected by the virus. This differential susceptibility is more pronounced in the presence of interferon. The tumor cell is characterized by having low levels, or no, PKR activity, or as being PKR−/−, STAT1−/− or both PKR−/− and STAT1−/−. The virus is selected from the group consisting of Rhabdovirus and picomavirus, and preferably is vesicular stomatitis virus (VSV) or a derivative thereof.

This application is a continuation of U.S. patent application Ser. No.10/743,639, filed Dec. 22, 2003 which is a divisional of U.S. patentapplication Ser. No. 09/664,444, filed Sep. 18, 2000, the contents ofwhich are incorporated herein by reference. This application claims thebenefit of U.S. Provisional Patent Application No. 60/287,590, having aneffective filing date of Sep. 17, 1999, the contents of which areincorporated herein by reference.

The present invention relates to a novel cancer therapeutic. Morespecifically, this invention relates to viruses that selectively infectand inhibit tumour cell growth.

BACKGROUND OF THE INVENTION

The use of oncolytic bacteria, or compositions of oncolytic bacterias,for combatting neoplasms in humans and animals is known. For example EP564 121, GB 1,587,244 and U.S. Pat. No. 3,192,116 disclose the use ofnon-pathogenic bacteria that result in the liquification and lysis oftumours in vertebrates. However in many instances, for example with theuse of Clostridium, the tumours are only partially destroyed, and tumourregrowth may still occur. To ensure control of tumour growth theadministration of bacteria, followed by chemotherapeutic drugs, forexample 5-fluorodeoxyuridine or alkylating agents, has been suggested(e.g. GB 1,069,144).

Several viruses have also been shown to exhibit tumoricidal properties,for example parvovirus H-1 (Dupressoir et al., 1996. Cancer Res,49:3203-3208), Newcastle disease virus (Reichand et al., 1992. J. Surg.Res, 52:448-453) or retroviral vectors containing drug susceptibilitygenes (Takamiya et al., 1993. J. Neurosurg, 79:104-110). WO97/26904 andWO96/03997 disclose a mutant herpes simplex virus (HSV-1761) thatinhibits tumour cell growth. Administration of HSV-1716 comprising a 759base pair deletion in each copy of γ34.5 of the long repeat region(R_(L)) to tumour cells kills these cells. However, this virus isspecific for neuronal cells as HSV is known to selectively inhabit theneuronal system. Furthermore, the use of common human pathogens as anoncolytic virus is limited as it is likely that the general populationhas been infected and acquired an immune response to such viruses. Apreexisting immune response to a viral strain similar to the one used asa therapeutic agent in the treatment of a cancer may attenuate theeffectiveness of the virus as therapeutic agent.

Other virus strains have reported oncolytic activity. The ONYX-015 humanadenovirus (produced by ONYX pharmaceuticals) is believed to replicatepreferentially in p53 negative tumour cells. This virus shows promise inclinical trials with head and neck cancer patients (Kim, D., T. et al.,Nat Med, 1998. 4:1341-1342). Reovirus type 3 is being developed byOncolytic Biotech as a cancer therapeutic, which preferentially grows inPKR−/− cells (Yin, H. S., J. Virol Methods, 1997. 67:93-101; Strong, J.E. and P. W. Lee., J Virol, 1996. 70:612-616; Strong, J. E., et al.,Virology, 1993. 197:405-411; Minuk, G. Y., et al., J Hepatol, 1987.5:8-13; Rozee, K. R., et al., Appl Environ Microbiol, 1978. 35:297-300).Reovirus, type III exhibited enhanced replication properties in cellswhich expressed the mutatnt ras oncogene (Coffey, M. C., et al.,Science, 1998. 282:1332-1334; Strong, J. E., et al., Embo J, 1998.17:3351-1362). Mundschau and Faller (Mundschau, L. J. and D. V. Faller,J Biol Chem, 1992. 267:23092-23098) have shown that the ras oncogeneproduct activated an inhibitor of PKR, and this coupled with theobservation that the PKR chemical inhibitor 2-aminopurine increased thegrowth of Reo type III in normal cells implicates PKR is a criticalregulator of the growth of reovirus.

W0 99/04026 teaches the use of VSV as a vector in gene therapy for theexpression of a wide range of products including antibodies, immunogens,toxins, etc. for the treatment of a variety of disease disorders.

Interferons are circulating factors which bind to cell surface receptorsactivating a signalling cascade ultimately leading to a number ofbiological responses. Two of the outcomes of interferon signalling aretightly linked: (1) an antiviral response and (2) induction of growthinhibitory and/or apoptotic signals.

U.S. Pat. No. 4,806,347 discloses the use of γ Interferon and a fragmentof INF-γ (known asΔ4α2) against human tumour cells.

WO 99/18799 reports the cytotoxic activity of Newcastle Disease Virus(NDV) and Sindbis virus towards several human cancer cells. However,both viruses demonstrated selectivity in their cytotoxic activitytowards tumor cells.

WO 99/18799 discloses that interferon addition to normal cells rendersthese cells resistant to NDV, yet, this effect was not observed withinterferon-treated tumor cells which continued to exhibit NDV-inducedsensitivity. WO 99/18799 also discloses the cytotoxic activity of VSVcells against KB cells (head and neck carcinoma) and HT 1080(Fibrosarcoma), and alleviation of cytotoxicity in normal and tumorcells, by VSV, in the presence of interferon. No other cell types weretested against VSV cytotoxic activity.

Certain mutant strains of VSV have been reported. Stanners, et al.,Virology (1987) 160(1):255-8. Francoeur, et al., Virology (1987)160(1):236-45. Stanners, et al., Gen. Virol. (1975) 29(3):281-96.Stanners, et al., Cell (1977) 11(2):273-81.

The present invention relates to viral formulations that are useful inthe treatment of diseases and cancers, preferably leukaemia. Suchformulations may also comprise an oncolytic VSV strain and a chemicalagent, for example a cytokine which confers to normal cells, resistanceto viral infection, but leaves diseased or cancerous cells susceptibleto viral infection and lysis.

It is an object of the invention to overcome disadvantages of the priorart.

The above object is met by the combinations of features of the mainclaims, the sub-claims disclose further advantageous embodiments of theinvention.

SUMMARY OF THE INVENTION

The present invention relates to a novel cancer therapeutic. Morespecifically, this invention relates to viruses that selectively infectand inhibit tumour cell growth.

According to the present invention there is provided a method ofreducing the viability of a tumour cell comprising administering a virusto the tumour cell, wherein the virus is characterized as not being acommon human pathogen. Preferably the tumour cell lacks PKR activity,and the virus is selected from the group consisting of rhabdovirus. Morepreferably the virus is VSV.

This invention is also directed to a method of reducing the viability ofa tumour cell comprising administering a virus to the tumour cell,wherein the virus is characterized as being unable to inactivate PKRactivity within a host cell. Preferably the virus is selected from thegroup consisting of vesicular stomatitis virus, picomavirus, influenzavirus, and adenovirus.

The present invention also pertains to a method of reducing theviability a tumour cell within a population of cells comprisingadministering a virus to the population of cells, wherein the virus ischaracterized as being able to selectively infect and kill the tumourcell. Preferably the virus is further characterized by being unable toinactivate PKR activity in a host cell.

This invention also relates to the method as defined above, wherein thepopulation of cells is treated with interferon prior to administeringthe virus.

This invention provides a method for identifying a tumor susceptible totreatment with a virus, comprising: (a) dividing a sample containingcells of the tumor into a first portion and a second portion; (b)treating the first portion with the virus; and (c)

-   -   determining whether the percentage of dead cells in the first        portion is higher than in the second portion, wherein the tumor        is susceptible to treatment with the virus if the percentage of        dead cells in the first portion is higher than in the second        portion.

This invention provides a method for identifying a tumor susceptible totreatment with a virus, comprising: (a) dividing a sample containingcells of the tumor into a first portion and a second portion; (b)treating the first portion with the virus and an amount of interferonsufficient to improve survival of interferon-responsive cells in thepresence of the virus, and treating the second portion with the virus inthe absence of interferon; and (c) determining whether the percentage ofdead cells in the first portion is higher than in the second portion,wherein the tumor is susceptible to treatment with the virus if thepercentage of dead cells in the first portion is higher than in thesecond portion.

The present invention is directed to a mutant VSV, characterized in thatthe mutant VSV grows poorly in interferon-responsive cells. Such strainsare also referred to herein as attenuated strains of VSV, or VSV strainsthat grow poorly in interferon-responsive cells. They can be identifiedby their producing smaller plaques in monolayers ofinterferon-responsive cells than in interferon-nonresponsive cells, asdescribed below. Attenuated VSV strains can also be identified by theirhaving a higher LD50 when administered intranasally to PKR+/− mice ascompared to WT Indiana, in the assay described below.

The present invention also pertains to a method for isolating VSV usingan affinity matrix, comprising adding the VSV to the affinity matrix toproduce bound VSV, washing the bound VSV, and eluting the VSV from theaffinity matrix. Also included in the present invention is a modifiedVSV that comprises a non-native fusion protein on the outer surface ofthe virus. The non-native protein may be fusion protein comprising anaffinity tag and a viral envelope protein, or it may be derived from aproducer cell.

The present invention is also directed to isolated nucleic acidmolecules (DNA or RNA) having a sequence coding for mutant VSV proteinsand sequences complementary thereto. Such nucleic acid molecules can beused in the preparation of a recombinant VSV or as a DNA vaccine.

There are several advantages for the use of a virus as described hereinas a therapeutic virus over other viruses:

-   Rhabdoviruses are not common human pathogens. For example, VSV is    found mostly in insects, rodents and domestic farm animals, and    therefore a large proportion of individuals will not have been    infected or immunized to VSV infection. On the other hand,    Adenovirus or Reovirus are human pathogens and most of the general    population have been infected and acquired an immune response to    both of these viruses. A preexisting immune response to a viral    strain similar to the one used as a therapeutic agent in the    treatment of a cancer may attenuate the effectiveness of the virus    as therapeutic agent;-   VSV replicates much more quickly than either Adenovirus or Reovirus,    and can be readily concentrated to high titres. Production of high    titre virus preparations is a significant limitation of other    potential viral therapeutic strains;-   VSV is simple virus comprising only five genes, easily amenable to    genetic manipulation. No such system is currently available for    Reovirus;-   Cellular infection by VSV is highly responsive to additional    chemical agents such as interferon, a feature which enhances its    therapeutic value.-   VSV has a broad host range and is capable of infecting most types of    human cells, whereas other viruses are more limited in regard to the    types of cells they may infect.-   VSV is a RNA virus and spends its entire lifecyle in the cytoplasm.    Therefore it involves less danger of unwanted integration into the    genome of a patient.    Collectively, these VSV attributes provide significant advantages    over the use of the other viruses known to exhibit oncolytic    activity.

This summary of the invention does not necessarily describe allnecessary features of the invention but that the invention may alsoreside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows a general schematic of the interferon cascade.

FIG. 2 shows the effect of VSV on normal human fibroblasts, humanmelanoma cell line SK-MEL3, LNCaP a prostate cancer cell line, and theovarian carcinoma cell A2780 in the presence and absence of interferonas determined by a modified cpe assay. Monolayers of cells were infectedat an moi of 0.1 pfu in a 12 well plate. At time 0 and every 12 hourssubsequent up to 48 hours, one well of infected cells was fixed with 0.5ml Leukostat fixative for 2 minutes. At the end of the experimentmonolayers were stained with Leukostat stains.

FIG. 3 shows the cytopathic effect of VSV in normal fibroblasts cells(FIG. 3 (a)), and tumour cell lines, including ovarian tumour cells(FIG. 3 (b)) and KB tumour cells (FIG. 3 (c)).

FIG. 4 shows the effect of VSV on normal human fibroblasts co-culturedwith 293T tumour cells over a period of 24 hours. Co-cultures wereinfected at an moi of 0.1 pfu/cell and the infection allowed to proceedin the presence (IFN+) or absence (IFN−) of interferon. Cultures werestained with antibodies to large T antigen (red nuclei) to detect the293T cells and with DAPI (blue nuclei) which stains all cell types.

FIG. 5 shows the effect of VSV in vivo on tumors implanted within nudemice.

Human melanoma cells were implanted within nude mice and either mockinjected (VSV(−)), injected with wild type VSV (data not presented), orinjected with additional melanoma cells infected in vitro with VSV forone hour prior to injection into the tumour site VSV(+)). Size of thetumors were determined over a 7 day period.

FIG. 6 shows ulcers formed on a tumor produced within a nude mouse asdescribed in FIG. 5.

FIG. 7: VSV and VSV infected cells inhibit growth of human melanomaxenografts in nude mice.

FIGS. 8A and 8B: PKR−/− mice are acutely sensitive to intranasal VSVinfection and demonstrate a deficiency in IFN mediated resistance.

FIG. 9: Interferon can protect xenograft bearing nude mice during VSVtreatment.

FIGS. 10A and 10B: Virus production from tumour cells and normal cellsinfected with wild type Indiana and various mutant VSV strains.

FIG. 11: Malignant cells are rapidly killed following VSV (WT Indiana)infection and are not protected by IFN-α.

FIG. 12: VSV induced cytopathic effect visible in human melanoma cellsbut not in primary human cells with or without IFN-α.

FIG. 13: Efficacy of a single intravenous dose of mutant VSV in treatinghuman melanoma xenografts in nude mice.

FIG. 14: N Protein cDNA sequence of wild type and mutant VSVs. GenBank Nnucl. (SEQ ID NO: 9); HR N nucl. (SEQ ID NO: 10); M2 N nucl. (SEQ ID NO:11); M3 N nucl. (SEQ ID NO: 12); M4 N nucl. (SEQ ID NO: 13).

FIG. 15: N Protein amino acid sequence of wild type and mutant VSVs.GenBank N a.a. (SEQ ID NO: 14); HR N a.a. (SEQ ID NO: 15); M3 N a.a.(SEQ ID NO: 16); M4 N a.a. (SEQ ID NO: 17).

FIG. 16: P Protein cDNA sequence of wild type and mutant VSVs. GenBank Pnucl. (SEQ ID NO: 18); HR P nucl. (SEQ ID NO: 19); M2 P nucl. (SEQ IDNO: 20); M3 P nucl. (SEQ ID NO: 21); M4 P nucl. (SEQ ID NO: 22).

FIG. 17: P Protein amino acid sequence of wild type and mutant VSVs.GenBank P a.a. (SEQ ID NO: 23); HR P a.a. (SEQ ID NO: 24); M2 P a.a.(SEQ ID NO: 25); M3 P a.a. (SEQ ID NO: 26); M4 N a.a. (SEQ ID NO: 27).

FIG. 18: M Protein cDNA sequence of wild type and mutant VSVs. GenBank Mnucl. (SEQ ID NO: 28); HR M nucl. (SEQ ID NO: 29); M3 M nucl. (SEQ IDNO: 30); M4M nucl. (SEQ ID NO: 31).

FIG. 19: M Protein amino acid sequence of wild type and mutant VSVs.GenBank M a.a. (SEQ ID NO: 32); HR M a.a. (SEQ ID NO: 33); M4 M a.a.(SEQ ID NO: 34); M3 M a.a. (SEQ ID NO: 35).

FIG. 20: G Protein cDNA sequence of wild type and mutant VSVs. GenBank Gnucl. (SEQ ID NO: 36); HR G nucl. (SEQ ID NO: 37); M2 G nucl. (SEQ IDNO: 38); M3 G nucl. (SEQ ID NO: 39); M4 G nucl. (SEQ ID NO: 40).

FIG. 21: G Protein amino acid sequence of wild type and mutant VSVs.GenBank G a.a. (SEQ ID NO: 41); HR G a.a. (SEQ ID NO: 42); M2 G a.a.(SEQ ID NO: 43); M3 G a.a. (SEQ ID NO: 44); M4 G a.a. (SEQ ID NO: 45).

FIG. 22: L Protein cDNA sequence of wild type and mutant VSVs. GenBank Lnucl. (SEQ ID NO: 46); HR L nucl. (SEQ ID NO: 47); M2 L nucl. (SEQ IDNO: 48); M4 L nucl. (SEQ ID NO: 49).

FIG. 23: L Protein amino acid sequence of wild type and mutant VSVs.GenBank L a.a. (SEQ ID NO: 50); HR L a.a. (SEQ ID NO: 51); M4 L a.a.(SEQ ID NO: 52).

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to a novel cancer therapeutic. Morespecifically, this invention relates to viruses that selectively infectand inhibit tumour cell growth.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

Cancer cells gain a survival advantage over their normal counterparts byacquiring mutations in growth inhibitory or apoptotic pathways and, inthe case of interferons, would do so at the expense of criticalantiviral defence mechanisms. As tumour cells gain a significant growthadvantage by mutating interferon response genes, they will be moresusceptible to virus infection.

By “reducing the viability” of a tumour cell it is meant either killingthe tumour cell or limiting its growth for a period of time.

By “not a common human pathogen” it is meant a virus that is foundmostly in non-human hosts, for example, but not limited to insects,rodents, and farm animals. Such viruses are not typically found withinthe general human population.

As used herein Mutant I, Mutant 1, Mut 1 and M1 refer to attenuatedmutant strain T 1026. Mutants II, III, IV and V (and variantnomenclature analogous to Mutant I) refer to attenuated mutants T1026R,TP3, TP6 and G31, respectively.

The novel cancer therapeutic of the present invention incorporates theuse of at least one oncolytic virus that selectively targets tumourcells and leads to their destruction. Preferably the oncolytic virus isa Vesicular stomatitis virus (VSV), for example the Indiana strain, orother strains, or a derivative thereof. By a derivative of VSV, it ismeant a VSV virus obtained by either selecting the virus under differentgrowth conditions, or one that has been subjected to a range ofselection pressures, or one that has been genetically modified usingrecombinant techniques known within the art. For example, which are notto be considered limiting in any manner, a derivative of VSV may includea mutant VSV selected following infection on a human cell that has beentreated with interferon as described herein, or a VSV that displays anaffinity tag useful for affinity purification.

The effectiveness of oncolytic virus suppression of tumour cell growthin part resides in the differential susceptibility of tumour cells,compared to normal cells, to viral infection. Without wishing to bebound by theory, the differential susceptibility may in part be due tothe down regulation or inactivation of factors within a cell thatotherwise function to protect the cell from tumorous growth and virusinfection. Examples of factors that when inactivated result in tumorouscell growth, and that are also involved in mediating virus infectioninclude but are not limited to PKR (double stranded RNA dependentkinase) and PML (Promyelocytic Leukemia gene), however, it is to beunderstood that other factors may also play a role.

The down regulation or inactivation of PKR, through a variety ofmechanisms including but not limited to PKR-related mediators, is knownto be associated with tumour cell growth, while normal cells exhibitactive PKR. Furthermore, wild type cells exposed to viral infectionexhibit elevated PKR expression which results in the suppression ofviral replication, while cells that exhibit reduced, or no, PKR activityare susceptible to viral attack and exhibit cancerous growth. Similarly,the PML gene product functions as a tumour suppressor and it is alsoknown to suppress viral replication.

By “differential susceptibility”, it is meant a property associated witha cell that results in both tumour cell growth and the inability of thecell to suppress viral replication. Cells exhibiting differentialsusceptibility are preferred candidates for treatment of tumorous cellgrowth using the cancer therapeutic of the present invention. Thisdifferential susceptibility may be accentuated through the addition ofone or more chemical agents prior to or during treatment of the tumourcell. Preferably, this chemical agent increases the resistance of awild-type cell to viral infection, but has little or no effect on theresponse of a tumour cell to viral infection. An example, which is notto be considered limiting in any manner, of such a chemical agent isinterferon.

By “PKR” it is meant a serine/threonine kinase that exhibits multiplefunctions including roles in the control of mRNA translation and genetranscription (1,2). The kinase harbors two double-stranded RNA dsRNAbinding motifs in its amino terminal regulatory half and catalytickinase domain in its carboxyl tail. Binding of dsRNA to the aminoterminus induces a conformational change in the enzyme revealing andactivating the catalytic kinase domain. The expression of PKR is inducedby several PKR-mediators, including but not limited to, interferon.

By “PKR-mediator” it is meant proteins or compounds that directly, orindirectly affect PKR activity either at the gene or protein level andinclude both PKR-activators and PKR-inhibitors. Examples ofPKR-activators include, but are not limited to STAT1 (see FIG. 1),Interferon regulatory factor (IRF-1), and interferon. Examples ofPKR-inhibitors, include, but are not limited to, VA RNAs, p58(IPK),factors associated with the Ras pathway, the ribosomal protein L18, orproteases that degrade PKR protein. PKR activity may also be mediatedthrough mutations to the gene encoding PKR, or to the regulatory regionthat drives the expression of PKR. These mutations may either increaseor decrease PKR activity. Mutations to PKR that reduce PKR activityinclude, but are not limited to, the loss of dsRNA binding ability ofPKR, or mutations that result in negative catalytic mutants. Mutationsthat increase PKR activity include, but are not limited toover-expression of PKR, or mutations that resulted in a more active PKRprotein.

PKR regulates translation through the phosphorylation of eIF-2α, afactor involved in the initiation of protein translation. Oncephosphorylated, eIF-2α-GDP, forms an inactive complex with eIF-2Bresulting in a rapid inhibition of protein synthesis. PKR impinges ongene transcription indirectly via activation of NFκB. This activationappears to be carried out by PKR phosphorylation of an IκB kinase (3)which in turn phosphorylates IκB leading to its targeted destruction.

PKR Antiviral Activity

Infection of a cell by many distinct virus types leads to the formationof dsRNA (e.g. as replicative intermediates) resulting in the activationof PKR and its subsequent downstream effectors (see FIG. 1). Inparticular, protein synthesis is rapidly terminated and an apoptoticcascade is initiated (4,5). As a result of the activation of PKR, theproduction of new virions is curtailed and the spread of virus throughthe organism is limited. Der et al (12) report a requirement for PKR inthe induction of cellular apoptosis in response to a variety of stressinducers.

Without being bound by theory, it is possible that malignancies arise asa result of multiple mutations in genes that control cell proliferationand apoptosis. PKR's role in regulating protein synthesis coupled withits antiproliferative and pro-apoptotic properties make it a target foroncogenic mutations, which directly or indirectly affect its activity.

As described in more detail in the examples an initial screen of severalviruses using PKR−/− animals indicated that PKR null animals aresusceptible to infection by Vesicular stomatitis virus (VSV). Similarresults were obtained in vitro, where VSV infection proceeded morerapidly in PKR−/− fibroblasts, when compared to infection in PKR+/+fibroblasts. These results demonstrate that PKR is required by mammaliancells to resist infections by VSV. Furthermore, certain cell lines, forexample, but not limited to primary human bone marrow, were resistant toVSV infection, while leukemia cell lines were susceptible to VSVinfection.

It is contemplated that viruses related to VSV, or other viruses thatexhibit similar mechanisms of viral infection can be identified thatexhibit the property of selectively infecting cells with reduced or noPKR activity. One of skill in the art can readily screen other virusesusing the methods as described herein, for their ability to reduce theviability of cells, or kill cells lacking PKR activity, or PKR−/− cells,PKR−/− animals, or both PKR−/− cells and animals.

As indicated in the examples below, pretreatment of cells withinterferon reduces virus infectivity by several orders of magnitude.Without wishing to be bound by theory, the addition of interferon mayupregulate PKR expression resulting in this increased resistance toviral infection.

Because of its potent antiviral activity, viruses have evolvedstrategies to circumvent PKR. For example, HIV and Hepatitis C encodeproteins dedicated to the binding and inactivation of PKR (6,7).Adenovirus encodes small RNA molecules (VA RNAs) which bind to but donot activate PKR (8). Influenza virus usurps a cellular protein p58(IPK)to inhibit PKR while polio virus initiates the proteolytic degradationof PKR (9,10). Large T antigen of SV-40 appears to function downstreamof eIF-2α to promote protein translation even in the presence ofactivated PKR (10).

PKR and Tumour Suppression

Expression of dominant negative PKR catalytic mutants in NIH 3T3 cellsleads to their malignant transformation and facilitates their growth astumours in nude mouse models (13,14). A similar phenomena has beenobserved using PKR mutants which have lost dsRNA binding activity.Induced expression of PKR in S. cerevisiae leads to growth arrest in theyeast—a phenomena that can be reversed by co-expression of anon-phosphorylatable version of eIF-2α. Therefore, PKR hasanti-proliferative activity and functions as a tumour suppressor.

There are several lines of evidence that PKR is inactivated, absent orreduced in expression in a broad spectrum of human malignancies:

-   Oncogenic Ras mutations occur in about 30% of all human tumours    while mutations in upstream Ras activators (ie EGF receptor, Neu    receptor, PDGF receptor) are even more common. Mundschau and Faller    (15,16) have described an oncogenic Ras induced PKR inhibitor.    Furthermore, Strong et al (17) demonstrated that activation of the    Ras pathway results in down regulation of PKR activity.-   The ribosomal protein L18 is overexpressed in primary colorectal    cancer tissues and has recently been shown to bind to and inactivate    PKR (18).-   Patients with 5q translocations exhibit diminished PKR expression    (19-21).

Interferon regulatory factor 1 (IRF-1) is a transcription factor withtumour suppressor activity, which maps to the human chromosomal region5q. PKR gene transcription is regulated in part by IRF-1.

-   Human PKR maps to ²p21-22 and has been recently identified as the    site of translocation in a case of acute myelogenous leukemia.-   In biopsies from poorly differentiated, highly malignant tumours,    PKR protein was present at very low levels or was undetectable    (23-25).    STAT1

STAT1 is an essential mediator of the interferon pathway and itsactivation results in an upregulation of PKR mRNA and protein (see FIG.1).

There is marked deficiency in the level/activity of STAT1 protein ininterferon resistant melanoma cell lines and primary melanoma biopsymaterial (26), in a variety of human tumour cell lines including amyeloid leukemia, cervical carcinomas, ovarian cancer, and a lungcarcinoma (27), and in a gastric adenocarcinoma (28,29). Furthermore,cutaneous T cell lymphoma (CTCL) is a malignancy which in general isresponsive to interferon (however frequently clinical resistance arisesin a substantial portion of cases). Sun et al (30) have reported thatSTAT1 protein is absent in a CTCL cell line suggesting that developmentof clinical resistance to interferon may arise due to STAT1 mutations.

PML: Promyelocytic Leukemia Gene

PML is an interferon induced gene that normally functions as a tumoursuppressor and a key regulator of Fas, TNFα and interferon inducedapoptosis. Recently, Chelbi-Alix et al have shown that another normalfunction of the PML gene product is to suppress virus replication. ThePML-RAR fusion protein functions as a dominant negative inhibitor ofinterferon induced apoptosis and we would predict will also make APLcells preferentially susceptible to virus infection.

Down regulation of PKR protein or activity occurs in a broad spectrum ofhuman malignancies. While cancer cells have attained a growth advantageand unbridled protein translation capacity by eliminating PKR orPKR-mediators, these cells have simultaneously eliminated one of thecell's primary and potent antiviral defence mechanisms. Therefore,tumour cells with reduced PKR activity will be more susceptible toinfection than their normal counterparts. As indicated above, othercomponents (e.g. STAT1 and PML) of the interferon pathway are frequentlymutated in human malignancies, and loss of their activity will rendertumour cells sensitive to virus infection. This differentialsusceptibility forms the basis for the use of viral-based cancertherapeutics of the present invention for the treatment of tumorous cellgrowth.

Screening of PKR null mouse strains with several different virusesindicated that PKR null animals are capable of suppressing a number ofvirus infections including vaccinia, influenza and EMCV. However,Vesicular Stomatitis Virus (VSV) exhibited an ability to infect PKR−/−animals. VSV, a member of the Rhabdovirus family, was observed to kill100% of PKR null animals following intranasal infection by as little as50 infectious virus particles (or plaque forming units, pfu). Incontrast, over 20,000 times as many VSV particles were required to killhalf of infected wild type littermates.

VSV is an enveloped, negative sense RNA virus with a simple five genegenome. This is a very well characterized virus family with severalserologically distinct laboratory strains and a multitude ofcharacterized mutants. The natural hosts of VSV include insects, rodentsand domestic farm animals. In general, very few North Americans havecome in contact with the virus—most human infections occurring inlaboratory personnel and farmers. In humans infections are eitherasymptomatic or manifested as mild “flu”. There are no reported cases ofsevere illness or death amongst infected humans.

The ability of VSV to selectively infect tumour cells over wild-typecells was also observed. Tumour cell lines, following an overnightinfection exhibited a 100 to 1000 times higher rate of infection thanthat detected in normal primary fibroblasts. Furthermore, the cytopathiceffect (cpe) was accelerated in the tumour cell cultures.

Since PKR is an interferon inducible gene product, pretreatment of cellswith interferon prior to exposure to VSV was tested to determined theeffect of viral infection. Wild-type cell cultures, that were pretreatedwith interferon, were resistant to VSV infection, while tumour celllines, for example, but not limited to, fibrosarcoma, melanoma, prostatecarcinoma, leukaemia and ovarian sarcoma, were susceptible to virusinfection (see Table 1, Example 2; FIG. 2). Lung carcinoma cells (LC80)were also susceptible to VSV infection in the presence and absence ofinterferon (data not presented). However, several tumour cell lines wereresistant to VSV infection in the presence of interferon.

Ovarian carcinoma cells, fibrosarcoma, lung carcinoma, melanoma,prostate carcinoma, lung carcinoma, and leukaemia cells are VSVsensitive, and this sensitivity was maintained in the presence ofinterferon, therefore, such tumor cells and cancers derived therefrommay be particularly amenable to VSV treatment. However, other cancersmay also be amenable to viral treatment as described herein. Studieswith respect to VSV sensitivity using primary tumour material is readilyavailable in ascites fluid. Further, since the tumour is containedwithin the peritoneal cavity it may prove particularly suited tolocalized administration of a virally based therapeutic. In this regard,live tissue from patient's ascitic fluid can be tested for the abilityof the tumour cells to support VSV infection in the presence and absenceof interferon.

It is expected that VSV will have therapeutic activity in vivo, and willhave the ability to kill distant (metastatic) tumour growths. To date nosignificant organ pathology in treated mice have been observed, however,the kinetics of VSV viremia need to be further studied. Nude mice,implanted with human melanoma cells received VSV, or additional melanomacells infected in vitro with VSV (see Example 5), to ensure thecontinuous production of infective particles to the tumour over aseveral hour period, via injection (FIG. 5). In mock-injected animals(VSV(−); injection with vehicle alone) tumours grew continuously overthe course of the experiment. Animals which received only pure virusshowed initially continuous growth of tumours over the first four dayperiod, after this time the tumours began to reduce in size andcontinued to do so over the course of this experiment. Tumours that wereinjected with infected cells stopped growing and regressed to small hardnodules resembling scar tissue. In some of the larger injected tumours,ulcers formed on the tumour within 1-2 days, (see FIG. 6). While bothinjection of purified virus and infected melanoma cells causedsignificant regressions, infected producer cells were more effective.

Studies with an immunocompetent mouse tumour model (i.e. as described byStrong et al; 17) will examine the affects of antibody response totherapeutic VSV infection, and determine if VSV infection of tumourcells increases their immunogenicity and promotes recognition of tumourantigens by the host organism.

Primary human bone marrow was also found to be resistant to VSVinfection in the absence of interferon pretreatment (see Table 1,Example 2), indicating that these cells have an innate resistance to VSVinfection. In contrast two leukemia cell lines (M07E and L1210) werealso tested and found to be susceptible to VSV infection as evidenced bycytopathic effect, virus growth and loss of cell viability.

While the results disclosed herein relate to VSV, it is to be understoodthat one of skill in the art, by following the methods outlined in thisdocument, will be readily able to screen other VSV strains, derivativesof VSV including mutants of VSV, or related viruses for the ability toselectively kill tumour cells. There are several other serologically andbiologically distinct strains of VSV, which can be tested for thisproperty. Such VSV strains include, but are not limited to New Jersey,Piry, Coccal, and Chandipura. Identification of other suitableserologically unrelated strains may be useful if sequential VSVinjections are required to completely eradicate tumours. Furthermore,picornaviruses (eg rhinoviruses) are known to be relatively innocuous tonormal human tissues yet grow extremely well in transformed cells intissue culture, and these viruses may also be used. Furthermore,combinations of viruses may be used to enhance the cytopathic effectobserved with VSV.

In order to determine whether the presence of either a normal or tumorcell could affect the other cell type (either normal and tumor cell) andalter the resistance or susceptibility of either of these cells to VSVinfection, normal cells and fibroblasts were co-cultured in the presentof VSV. The culture was infected at an moi of 0.1 pfu/cell and theinfection allowed to proceed in the presence or absence of interferon.At 0, 12 and 24 hours (FIG. 4) the cultures were fixed and stained withantibodies to large T antigen (red nuclei) to detect the 293T cells andwith DAPI (blue nuclei) which stains all cell types (FIG. 4). The numberof 293T cells (red nuclei) steadily declined during the time course anddisplayed severely condensed or fragmented nuclei characteristic of acell dying from virally induced apoptosis. This selective destruction ofthe transformed cells was seen both in the presence and absence ofinterferon. The normal fibroblasts did not develop nuclear changes norwere their numbers reduced in response to VSV infection even though 293Tcells were producing copious amounts of virus within the co-culture.This indicates that mixtures of cell populations may be treated with VSVwhile still maintaining tumor cell sensitivity, and normal cellresistance to VSV.

In addition there are a number of mutants of VSV, for example, but notlimited to mutants which are impaired in the shut down of host proteinsynthesis or are more or less sensitive to interferon, which may exhibitdifferential infection between normal and tumour cells. For example,which is not intended to be limiting in any manner, other viral mutantsare known which show tropism for STAT1 or PKR negative cells include aninfluenza virus strain which is unable to inactivate PKR has beendescribed (36) Adenovirus mutants which lack the PKR inactivating VAgene are known to grow better in the absence of PKR.

As described herein, VSV mutants were isolated that grew poorly oninterferon responsive cells. These mutants were selected based upontheir ability to form small plaques in monolayers ofinterferon-responsive cells. On interferon non-responsive cells (i.e.tumor cells) these mutants form large plaques. The selection of mutantsby size of plaque in interferon-responsive cells allows for theisolation of virus that grows poorly in normal cells. However, other VSVmutants may be obtained under different selection criteria. Mutantsisolated using interferon-responsive cells were amplified and tested fortheir ability to kill tumour and normal cells. The rationale here isthat VSV mutants, which can induce interferon in target cells, wouldlimit their own replication in an interferon responsive cell population.These same viruses would however have unrestricted growth in tumourcells that lack interferon responsiveness. These mutants are of value,as they have even less cytopathic effect on normal tissues whilemaintaining oncolytic activity than wild type VSV.

Four mutants (Mut 1-4) were obtained based on their ability to formplaques in monolayers of interferon-responsive cells. These mutants, andwild type virus (moi of 1.0 pfu/cell) were used to infect melanoma cellsand normal human foreskin fibroblasts. All of the mutants were able tokill tumour cells efficiently but normal cells infected with the mutantseven after long periods of infection appeared completely uninfected. Atthis same moi wild type VSV demonstrated a cytopathic effect on thenormal cells. These results indicate that the mutant virus have agreater therapeutic effect in that they kill tumour cells efficientlywhile sparing normal cells, and that they also have the ability toproduce more virus particles and increase virus spread throughout thetumour (see Example 4). Surprisingly these mutants grew more rapidlythan wild type VSV (Indiana) in HCT 116 colon carcinoma cells but not inOSF7 cells (See Example 21 and FIGS. 10A and 10B). VSV mutants thatdisplay rapid growth in the tumour cell of interest but not in normalcells are preferred.

Earlier experiments indicated that PKR−/− mice were killed with VSV byseveral routes of infection, however, these mice were not affected byintravenous injections of the virus. In order to determine whetherplasma components were inactivating the virus upon contact, VSV producedfrom several sources including within mouse L cells was incubated withhuman serum (from normal uninfected donor) and the virus titer afterincubation determined (Example 6). The viral titer of L cell-producedVSV dropped four hundred fold, while VSV produced in human melanomacells was unaffected by incubation in plasma. These results indicatethat the choice of cell line for the production of VSV is critical.Based on this observation it is possible to screen human cell lines forthose that produce optimum amounts of virus that is not sensitive tohuman serum.

Without wishing to be bound by theory, it may be that the difference inthese two virus preparations reflects the nature of the cohort ofproteins found on the surface of the virus producing cells. As part ofits replicative cycle, VSV buds through the plasma membrane and acquirescellular protein on its envelope. Certain proteins found on L cells whenexpressed in the context of the virus particle could activatecomplement. Indeed, it has been shown earlier that retrovirus particlesproduced in certain mouse cells are inactivated by serum while the samevirus produced in a subset of human cell lines was unaffected by plasma.(Pensiero, M. N., et al. Hum Gene Ther, 1996. 7:1095-1101).

Conventional techniques for VSV production are difficult to scale up forindustrial production. Therefore, the purification of VSV, using anaffinity matrix, for example affinity chromatography was explored. (SeeExample 7). However, other protocols for affinity purification may alsobe used as known within the art, for example, but not limited to, batchprocessing a solution of virus and affinity matrix, pelleting theVSV-bound matrix by centrifugation, and isolating the virus. In order toprovide the virus with an affinity tag to be used for the purificationof the virus, the virus may be genetically modified, using techniqueswell known in the art, to express one or more affinity tags on itssurface, preferably as a fusion viral envelope protein, or producer celllines may be engineered to express one or more affinity tags on theirplasma membranes which would be acquired by the virus as it buds throughmembrane, however, endogenous viral envelope proteins may also be used.One well characterized affinity tag involve the use of Histidineresidues which binds to immobilized nickel columns, however, it is to beunderstood that other affinity tags may also be employed.

Cell lines can be prepared that act as a universal producer of VSV, orother virus, that expresses a chimeric VSV protein with nickel binding,or other affinity tag properties. This universal producer cell may beused for the production of a chimeric protein (affinity tag) for anyenveloped virus (including all enveloped RNA and DNA viruses). For thepurification of virus which bud through the nuclear membrane (such asHerpes virus), a tag to be expressed on the viral envelope proteinexpressed in the nuclear membrane is engineered.

Other affinity tags include antibodies, preferably an antibody whichrecognizes a particular peptide under conditions of low salt, lowtemperature or in the presence of a critical cation/anion. Physiologicalsalt concentrations, thermal elution or chelation could effect elution.Antibodies generated against di or tripeptides may also be used forpurification. In this manner, two or more of these tags on the surfaceof a single virus particle would allow for the sequential affinitypurification of the virus.

VSV may be genetically modified in order alter its properties for use invivo. Methods for the genetic modification of VSV are well establishedwithin the art. For example a reverse genetic system has beenestablished for VSV (Roberts A. and J. K. Rose, Virology, 1998. 247:1-6)making it possible to alter the genetic properties of the virus.Furthermore, standard techniques well known to one of skill in the artmay be used to genetically modify VSV and introduce desired genes withinthe VSV genome to produce recombinant VSVs (e.g. Sambrook et al., 1989,A Laboratory Manual. New York: Cold Spring Harbor Laboratory Press).

VSV may be targeted to a desired site in vivo to increase viralefficacy. For example, modification of VSV G protein to produce fusionsthat target specific sites may be used to enhance VSV efficiency invivo. However, it is to be understood that other protein targets inaddition to the VSV G protein may also be modified to produce suchfusion proteins. Such fusion proteins may comprise, for example, but notlimited to, Single chain Fv fragments (Lorimer, I. A., et al. Proc.Natl. Acad. Sci. U.S.A., 1996. 93:14815-20) that have specificity fortumour antigens. An example of such a single chain Fv fragment that maybe used to prepared a VSV G fusion protein, is an Fv fragment thattargets a mutant EGF receptor found on about 80% of human breast tumourcells.

VSV may also be modified to express one or more suicide genes capable ofmetabolizing a pro-drug into a toxic metabolite thereby permitting VSVinfected cells to be killed by administration of a pro-drug. Forexample, VSV comprising the herpes virus thymidine kinase gene or thecytosine deaminase gene encodes an enzyme that can convert gancicloviror 5-FC, respectively, into a toxic compound. However, it is to beunderstood that other suicide genes may also be employed. As it is wellestablished that ganciclovir metabolites kill not only cell expressingHSV TK but also cells in the immediate vicinity, rVSV comprising thesesuicide genes exhibit several advantages. For example, the effectivekilling by the virus is increased since one infected cell kills ten ormore surrounding tumour cells, furthermore rVSV comprising a suicidegene permits the elimination of virus if desired from an individualinfected with the virus. This may be important in situations where it isunclear how VSV may affect an individual. For instance, an immunecomprised individual may be unexpectedly susceptible to VSV. Thus theaddition of a suicide gene would be an improvement on the safety of theviral therapeutic.

VSV may also be modified by the introduction of a mammalian geneproduct. Such a mammalian gene product would limit VSV growth in normalcells, but not the growth of VSV in tumour or diseased cells. Forexample, rVSV capable of expressing one or more transactivators of p53,activates apoptotic pathways in normal cells but not tumor cells. SuchrVSVs therefore selectively limit virus spread in normal tissues.However, it is to be understood that other mammalian gene products mayalso be expressed within VSV for this purpose. Another example, which isnot to be considered limiting in any manner is the PKR gene. A rVSVexpressing the PKR gene limits virus replication in all normal cells,however, in cells that express PKR inhibitors, the virally encoded PKRis inactivated. An example of a cell that expresses one or more PKRinhibitors is a chronically Hepatitis C infected cell. Since Hepatitis Cencodes and expresses two known inhibitors of PKR (i.e. NS5A and E2), aVSV encoded PKR gene product is be neutralized, and VSV allowed toreplicate freely.

The above description is not intended to limit the claimed invention inany manner, furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

The present invention will be further illustrated in the followingexamples. However it is to be understood that these examples are forillustrative purposes only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLE 1 PKR negative Cells are Susceptible to VSV Infection

In vivo Experiments

Initial studies were directed to identifying viruses that are capable ofinfecting PKR−/− animals and cells. Using homologous recombinationstrategies, PKR null mouse strains were generated (35, which isincorporated by reference) and tested for their ability to fight virusinfections. Since these mice are PKR−/−, they should be susceptible tovirus infection. Several species of virus were administered to PKR nullanimals over a range of concentrations.

Infection of PKR Null Mice:

A PKR null mouse line was generated using conventional knockouttechnology (Abraham, N., et al., J Biol Chem, 1999. 274:5953-5962.).Groups of five female mice, 3 months of age or greater, were infectedintranasally with varying amounts of vesicular stomatitis virus (Indianastrain). Age matched wild type animals were infected in parallel andboth sets of animals were monitored on a daily basis for signs ofinfection. These include, hydration, piloerection, activity level,appetite, hind limb paralysis, respiratory rate, body weight and anyother symptoms indicating that the animal was in distress.

Wild type animals showed few and only transient symptoms atmultiplicities of infection up to 10⁵ pfu with VSV. In contrast, PKRnull animals very rapidly developed dehydration, piloerection, loss ofappetite, rapid respiratory rate, decreased activity and squintingcrusty eyes. At high doses of VSV infection (10⁵ pfu) the animals showedsymptoms in less than 24 hours and usually succumbed to the infectionwithin 48 hours. At doses of infection as low as 25 plu, 100 percent ofthe PKR null animals died of VSV infection within 5 days. In separateexperiments groups of five wild type and PKR null animals weresacrificed at 48 hours post infection with VSV and organs were removedto assess viral titres. In the PKR animals titres in excess of onemillion PFU/ml of lung homogenate were found at this time while in wildtype animals virus titres ranged from 0 to 100 pfu per ml of lunghomogenate. In the wild type and PKR null animals similar amounts ofvirus were found in the brain. The remainder of the tissues in bothmouse strains had undetectable virus at this time post infection.

Vesicular stomatitis virus, a member of the Rhabdovirus family was ableto kill 100% of PKR null animals following intranasal infection by aslittle as 50 infectious virus particles (or plaque forming units, pfu).In contrast, over 20,000 times as many VSV particles were required tokill half of infected normal littermates. These results indicate thatPKR null animals are capable of suppressing a number of virus infectionsincluding vaccinia, influenza and EMCV. However, VSV exhibited anability to infect PKR−/− animals. These results also indicate that PKRis required by mammalian cells to resist infections by VSV (Indianalaboratory strain).

EXAMPLE 2 Selective Killing of Tumour Cells with VSV

In vitro Experiments

Several tumour cell lines were chosen at random from the Ottawa RegionalCancer Center and tested for their susceptibility to VSV infection.Primary fibroblast cultures from healthy adult volunteers or primarybone marrow samples from healthy donors were used as control cells.

Infection of Tumour Cells with VSV:

As a first test of the oncolytic properties of VSV, virus production andcytopathic effect following an overnight incubation with VSV wasassessed. Monolayers of cells were incubated with the Indiana strain ofVSV at a multiplicity of infection (moi) of 0.1 plaque forming units(pfu). After allowing virus to adsorb for 30 minutes at 37 C, thecultures were rinsed thoroughly with phosphate buffered saline (PBS) andthen cultured an additional 18 hours at 37 C. At this time, the cultureswere examined microscopically for cytopathic effect (cpe) andphotographed. The 18 hour supernatant was removed and virus titres perml of medium determined. In some experiments, cultures were preincubatedfor 12 hours with human alpha interferon (100 units/ml) prior toinfection.

To examine the kinetics of infection of the assorted cell types amodified cpe assay (Heise, C., et al., Nat Med, 1997. 3:639-645) wasused. Essentially, monolayers of cells were infected at an moi of 0.1pfu in a 12 well plate. At time 0 and every 12 hours subsequent up to 48hours, one well of infected cells was fixed with 0.5 ml Leukostatfixative (Fisher Diagnostics) for 2 minutes. At the end of theexperiment monolayers were stained with Leukostat stains 1 and 2following manufacturers instructions. Since PKR is an interferoninducible gene product, the pretreatment with interferon, 100 units/mlof human alpha interferon 12 hours prior to infection, was tested todetermined if interferon could enhance protection within the assortedcell cultures. The data are presented in Table 1 and FIGS. 2-3. TABLE 1Cell lines tested for VSV sensitivity Untreated Overnight InterferonRefer- Virus Overnight Cell line cell type ence Yield Virus Yield OSF 16human normal fibro- ORC 1 × 10⁵ pfu 0 pfu blast C¹ AG1522 human foreskinfibro- [20] 0 pfu blast OSF 7 human normal fibro- ORC 1 × 10⁶ 0 pfublast C OSF 12 human normal fibro- ORC 2 × 10⁵ pfu 0 pfu blast C MN11mouse fibrosarcoma [21] 1 × 10⁸ 1 × 10⁴ A2780 human ovarian [22] 2 × 10⁸1 × 10⁷ carcinoma H-1078 normal human bone ORC 0 pfu (moi Not marrow C10 pfu) determined MO7E human leukemic cell [23] 2 × 10⁶ Not line (moi1.0 pfu) determined L1210 mouse leukemic cell [24] 4 × 10⁶ 2 × 10⁴ lineSK-MEL3 human melanoma [25] Not Not determined: determined: cpe assaycpe assay positive positive LNCAP human prostate [26] Not Not carcinomadetermined: determined; cpe assay cpe assay positive positive 293Tfibrosarcoma trans- [27] 1 × 10⁸ 8 × 10⁷ formed with SV-40 Large T andAdeno E1A OVCA [28] 1 × 10⁷ 0 pfu 432 C13 ovarian carcinoma [29] 1 × 10⁸1 × 10⁵ OVCA 3 [30] 5 × 10⁷ Not determined COS Large T transformed [31]2 × 10⁸ Not simian kidney cell line determined HCT 116 colon carcinoma[32] Not Not determined: determined cpe assay cpe assay positivepositive OVCA [28] 1 × 10⁸ 3 × 10⁶ 420¹established at the ORCC from forearm biopsy.

From the data in Table 1 it can be seen that although normal humanfibroblasts can support viral replication, the amount of virus producedand the progression to cell lysis was substantially delayed whencompared to tumour cells. An even more substantial difference in virusproduction was observed following pretreatment with interferon. Whilenormal human fibroblast monolayers were completely protected from thecytolytic affect of VSV by interferon, tumour cells remained sensitive,producing copious amounts of viral particles and rapidly undergoingcytolysis.

Other cells lines, inlcuding a lung carcinoma cell line (LC80) and aleukaemia cell line, AML5 (acute myelogenous leukemia 5) cells were alsofound to be effectively killed by VSV. In the case of AML5, at a moi of1.0 pfu/ml cells were completely killed within 24 hours, while at 0.0001pfu/ml the cells were killed within 72 hours, further indicating thesensitivity of leukaemia cells to VSV.

As can be seen in FIG. 2, monolayers of tumour cells were much morerapidly destroyed by VSV infection as compared to normal humanfibroblasts. The human melanoma cell line SK-MEL3, the LNCaP prostatecancer cell line and the ovarian carcinoma cell A2780 all showedsubstantial cpe as early as 12 hours post infection.

Although the normal human fibroblast cultures were infected and capableof producing virus (see Table 1), the kinetics of infection wassubstantially slower than in the three tumour cell lines tested in thisexperiment. In addition, as with the overnight virus growth assay (Table1, FIG. 2), interferon alpha treatment completely protected the normalhuman fibroblasts, but was ineffective at protecting the three tumorcell lines from the cytopathic effect of VSV.

The results obtained for Table 1 demonstrate that a screening strategyfor determining the types of tumours which are susceptible to killing byVSV may be employed using for example, but not limited to, the NIH/NCIstandard panel of tumour cell lines available from ATCC. These celllines are screened in order to determine the time to complete cpe and/orvirus growth using various initial multiplicities of infection. Theseexperiments are done in the presence and absence of interferon so thatthe number of and types of tumours that are VSV sensitive and areresistant to interferon's antiviral activity are determined.

VSV Treatment of Leukemia

VSV does not productively infect bone marrow stem cells , even at highmoi of 10 pfu/cell (H-1078; Table 1). The treated cultures retained allof their stem cell characteristics. Two leukemia cell lines (MO7E andL1210; Table 1) were killed following an overnight infection andproduced large amounts of virus.

To determine whether VSV could kill primary leukemia cells from a cancerpatient, a peripheral blood sample was obtained from an AML patient andwhite blood cells collected and plated in RPMI media plus 10% FBS(10⁷/well in 6 well plate, each infection in duplicate). Cells were mockinfected or infected at an moi of 10.0/cell. VSV selectively killedmyeloid leukemic cells as indicated by the decrease in the percentage ofblast cells (leukemic blasts), while the overall cell number wasminimally affected (i.e. neutrophils flourished). The leukemic sampleproduced titres of VSV exceeding 10⁷ pfu/ml at 16 hours post infection.The number of blast cells in the sample was dramatically reduced at 21hours post infection while the proportion of normal neutrophilsincreased. Mock infected cells (−VSV) contained almost 70% blast cellsin a monolayer, while in cells infected with VSV (+VSV) normal cellspredominated. These results demonstrate VSV is able to preferentiallykill primary leukemic blast cells while sparing normal blood cells.

EXAMPLE 3 Killing of Tumour Cells in Mixed Cultures

Normal human fibroblasts and 293T tumour cells were co-cultured in a50:50 mixture. Since 293T cells express the large T antigen which is notfound in normal cells, the two cell types can be distinguished byimmunofluoresence.

In this experiment cultures were infected at an moi of 0.1 pfu/cell andthe infection allowed to proceed in the presence or absence ofinterferon. At 0, 18 and 24 hours (FIG. 4) the cultures were fixed andstained with antibodies to large T antigen (red nuclei) to detect the293T cells and with DAPI (blue nuclei) which stains all cell types (FIG.4). Initially both cell types displayed a spindle-like morphology withlarge oval nuclei. After 18 hours the number of 293T cells (red nuclei)were reduced and many of the remaining 293T cells displayed alterednuclear morphology. By 24 hours post-infection very few 293T cells weredetected and those few that remained displayed severely condensed orfragmented nuclei characteristic of a cell dying from virally inducedapoptosis.

This selective destruction of the transformed cells was seen both in thepresence and absence of interferon. The normal fibroblasts did notdevelop nuclear changes nor were their numbers reduced in response toVSV infection even though 293T cells were producing copious amounts ofvirus within the co-culture.

EXAMPLE 4 VSV Mutants as Oncolytic Agents

VSV mutants were isolated based upon their ability to form small plaquesin monolayers of interferon-responsive cells, as compared to the size ofplaques in monolayers of interferon-nonresponsive cells. Viral isolates,which form small plaques in interferon-responsive cells were picked,amplified and re-cloned. Mutants isolated in this way were amplified andtested for their ability to kill tumour and normal cells. The rationalehere is that VSV mutants, which can induce interferon in target cells,would limit their own replication in an interferon responsive cellpopulation. These same viruses would however have unrestricted growth intumour cells that lack interferon responsiveness. These mutants would beof value, as they should have even less cytopathic effect on normaltissues while maintaining oncolytic activity.

Four mutants (Mut 1-4) were obtained based on their ability to formsmall plaques in monolayers of interferon-responsive cells. Thesemutants were initially identified by Dr. Lauren Poliquin (University ofQuebec at Montreal) and provided by him. After five rounds of plaquepurification, these mutants and wild type virus (moi of 1.0 pfu/cell)were used to infect melanoma cells and normal human foreskin fibroblastsand titres of released virus determined 12 and 24 hours post infection.

All of the mutants were able to kill tumour cells efficiently but normalcells infected with the mutants even after long time points appearedcompletely uninfected. At this same moi wild type VSV demonstrated acytopathic effect on the normal cells. It was also observed that all ofthe VSV mutants produced approximately ten times more virus than thewild type VSV following an overnight infection of melanoma cells. Onnormal cells, while the Mutants 1-4 had significantly less cytopathiceffect than wild type VSV, similar amounts of virus were produced fromthe infected cultures. These results indicate that the mutant virus havea greater therapeutic effect in that they kill tumour cells efficientlywhile sparing normal cells, and that they also have the ability toproduce more virus particles and increase virus spread throughout thetumour.

EXAMPLE 5 Infection of Nude Mice Bearing Human Tumour Xenografts

Nude mice were implanted with human melanoma cells and divided intogroups. One group received a mock injection (VSV(−)), and the other wereinjected with wild type VSV or injected with additional melanoma cellsinfected in vitro with VSV for one hour prior to injection into thetumour site in order to deliver cells that would continuously produceinfective particles to the tumour over a several hour period (VSV(+)).The results of these experiments are seen in FIG. 5 which shows theaverage of the tumour area with time in treated and mock injectedanimals.

In the case of mock-injected animals (VSV(−); injection with vehiclealone) tumours grew continuously over the course of the experiment.Animals which received only pure virus showed initially continuousgrowth of tumours although at day 4 post infection the tumours began toshrink and continued to do so over the course of this experiment.Tumours that were injected with infected cells demonstrated the mostdramatic regressions. Essentially most tumours stopped growing andregressed to small hard nodules resembling scar tissue.

In some of the larger injected tumours, ulcers formed on the tumourwithin 1-2 days, (see FIG. 6), followed by continuous shrinkage of theonce rapidly growing malignancy. While both injection of purified virusand infected melanoma cells caused significant regressions, infectedproducer cells were more effective.

EXAMPLE 6 The Choice of Cell Line for Producing VSV Affects Sensitivityof the Virus to Plasma

Earlier experiments indicated that PKR−/− mice were killed with VSV byseveral routes of infection, however, these mice were not affected byintravenous injections of the virus. Without wishing to be bound bytheory, this could be because the PKR−/− vascular endothelial cellsprovide a barrier to tissue infection or because plasma components wereinactivating the virus upon contact. To test this latter idea VSVproduced from several sources including within mouse L cells wasincubated with human serum (from normal uninfected donor) and the virustiter after incubation determined.

Following incubation of VSV in human serum, the viral titer of Lcell-produced VSV dropped four hundred fold. On the on the other handVSV produced in human melanoma cells was unaffected by incubation inplasma.

These results indicate that the choice of cell line for the productionof VSV is critical. Based on this observation it is possible to screenhuman cell lines for those that produce optimum amounts of virus that isnot sensitive to human serum.

EXAMPLE 7 Strategy for VSV Concentration and Purification

Conventional techniques for VSV production include centrifugation stepsand gradient purification—both of these approaches difficult to scale upfor industrial production. Therefore, alternate protocols for thepurification of VSV, for example affinity columns for the simultaneousconcentration and purification of virus particles has been explored.

In order to provide the virus with an affinity tag to be used for thepurification of the virus, endogenous proteins may be used or, the virusmay be engineered to express one or more affinity tags on its surface,or producer cell lines may be engineered to express one or more affinitytags on their plasma membranes which would be acquired by the virus asit buds through membrane. The unique viral envelope proteins can bepurified using affinity chromatography.

One such affinity tag may involve the use of Histidine residues whichbinds to immobilized nickel columns, however, it is to be understoodthat other affinity tags may also be employed. This approach has beentested using the bacterial virus M13. Using a phage peptide displaysystem (Koivunen, E., et al., J. Nucl Med, 1999. 40:883-888), viralparticles expressing Histidine containing peptides which bind to nickelcolumns, but that can be eluted with imidazole, were selected including:

-   CTTHRHHTSNC (SEQ ID NO:1); CLNAHRTTHHHC (SEQ ID NO:2); CHGLHSNMRHC    (SEQ ID NO:3); CHHHHRLNC (SEQ ID NO:4); CHSHHHRGC (SEQ ID NO:5);    CWDHHNHHC (SEQ ID NO:6); CDNNHHHHC (SEQ ID NO:7); CHHHRISSHC (SEQ ID    NO:8). The expression of these peptides on the surface of M13 phage    resulted in the purification concentration of the virus on nickel    resins and their elution using low concentrations of imidazole.

One or more of these sequences can be integrated into the VSV G proteinto result in an increased concentration of the viral particles bearingthese peptides on nickel residues. The eluted virus is expected toretain its infectivity.

In this manner a cell line that can be a universal producer of VSV, orother virus, that expresses a chimeric VSV protein with nickel bindingproperties is produced. This universal producer cell may be used for theproduction of such a chimeric protein (affinity tag) for any envelopedvirus (including all enveloped RNA and DNA viruses). For thepurification of virus which bud through the nuclear membrane (such asHerpes virus), a tag to be expressed on the viral envelope proteinexpressed in the nuclear membrane is engineered.

Other affinity tags include antibodies, preferably an antibody whichrecognizes a particular peptide under conditions of low salt, lowtemperature or in the presence of a critical cation/anion. Physiologicalsalt concentrations, thermal elution or chelation could effect elution.Antibodies generated against di or tripeptides may also be used for forpurification. In this manner, two or more of these tags on the surfaceof a single virus particle would allow for the sequential affinitypurification of the virus.

EXAMPLE 8 Use of VSV to Treat Chronic Infections

Some human disorders arise as a result of chronic viral infectionsincluding latent herpes infection, hepatitis, AIDS and cervical cancer.In each of these cases, the causative viral agent has evolved mechanismsto inactivate components of the interferon response pathway includingPKR (e.g. Chelbi-Alix, M. K. and H. de The, Oncogene, 1999. 18:935-941;.Gale, M. J., Jr., et al., Virology, 1997. 230: 217-227; Gale, M. J., etal., Clin Diagn Virol, 1998. 10:157-162; Gale, M., Jr. and M. G. Katze,Methods, 1997. 11:383-401; Barnard, P. and N. A. McMillan, Virology,1999. 259:305-313). Therefore, the administration of VSV, or interferoninducing VSV mutants, or a combination thereof, to individuals sufferingfrom these disorders, selectively ablates the chronically infectedcells. Further therapeutic efficacy could be found by targeting throughcell or viral receptors only the chronically infected cells.

EXAMPLE 9 Genetic Modification of VSV

A reverse genetic system has been established for VSV (Roberts A. and J.K. Rose, Virology, 1998. 247:1-6) making it possible to alter thegenetic properties of the virus.

Targeting VSV to Desired Sites In vivo

Presently VSV can bind to most mammalian cell types although itsreplication once inside the cell can be restricted (i.e. by interferonresponsive gene products including PKR). Thus the effective dose ofvirus that can actually find target cells (i.e. tumour cells) forproductive infection can be greatly limited simply by the “sink” thatother normal tissues provide. Therefore, VSV may be genetically modifiedin order to bind and infect only tumour cells.

Recombinant DNA techniques well known in the art (e.g. Sambrook et al.,1989, A Laboratory Manual. New York: Cold Spring Harbor LaboratoryPress) are used to modify VSV G protein. Single chain Fv fragments(Lorimer, I. A., et al. Proc. Natl. Acad. Sci. U.S.A., 1996.93:14815-20) that have specificity for tumour antigens are fused to VSVG protein. An example of such a single chain Fv fragment is one thattargets the mutant EGF receptor that is found on about 80% of humanbreast tumour cells.

Expression of Suicide Genes within VSV

The VSV genome is modified so that it comprises the herpes virusthymidine kinase gene or the cytosine deaminase gene. Both of thesegenes encode enzymes which can convert pro-drugs into toxic compounds(e.g. ganciclovir or 5-FC). Viruses modified in this way express thesesuicide genes, thereby permitting VSV infected cells to be killed byadministration of the pro-drug. This provides two advantages since(1) itis well established that ganciclovir metabolites kill not only cellexpressing HSV TK but also can cells in the immediate vicinity. This“by-stander effect” can increase the effective killing by the virus(i.e. one infected cell could result in the killing of ten or moresurrounding tumour cells); and (2) having a VSV with a suicide genecould allow the elimination of virus if desired from an individualinfected with the virus.

Controlling VSV Growth In Vivo

A mammalian gene product is introduced within VSV to limit VSV growth innormal cells, but this gene product does not affect VSV growth in tumouror diseased cells.

Recombinant VSVs (rVSV) comprising one or more transactivators of p53,activate apoptotic pathways in normal cells but not tumour cells. SuchrVSVs limit virus spread in normal tissues but allow virus growth intumour cells.

rVSV comprising the PKR gene limits virus replication in all normalcells, however, in cells that express PKR inhibitors, the virallyencoded PKR is inactivated. An example of a cell that expresses one ormore PKR inhibitors is a chronically Hepatitis C infected cell. SinceHepatitis C encodes and expresses two known inhibitors of PKR (i.e. NS5Aand E2), a VSV encoded PKR gene product is be neutralized, and VSVallowed to replicate freely.

EXAMPLE 10 Progressive Loss of Interferon Responsiveness with OncogenicTransformation

Murine fibroblasts at various stages of transformation, eitherpretreated with 100 units of interferon alpha or left untreated, wereinfected with WT Indiana VSV at an MOI of 0.1 pfu/cell. Viral productionwas measured 18 hours pi by standard plaque assay. MEF: mouse fibroblastprimary cultures isolated from Balb/C mouse embryos. NIH 3T3 cells:immortalized mouse embryo fibroblasts. PVSrc: NIH 3T3 cells transformedwith the viral src gene. MOP 8: NIH 3T3 cells transformed with thepolyoma virus Large T antigen. Results are shown in Table 2.

In this example, loss of interferon responsiveness correlates withsusceptibility to VSV infection and progression of the malignantphenotype. The MEF cells are mortal (ie have a limited lifespan inculture) and completely interferon responsive. NIH 3T3 cells althoughnot tumourigenic are immortalized and are about ten thousand fold lessresponsive to interferon than MEFs. The PVSrc and MOP 8 cells are fullytumourigenic, support robust VSV replication and are minimally protectedby interferon treatment. TABLE 2 Viral Titre (pfu/ml) Cell LineUntreated IFN-α MEF (Mouse Embryonic Fibroblast) 4 × 10⁶ <10 NIH3T3 8 ×10⁷ 1 × 10⁴ PVSrc 3 × 10⁹ 2 × 10⁷ MOP 8 1 × 10⁸ 5 × 10⁶

EXAMPLE 11 Virus Yield After Overnight Infection of Various Cell LinesEither Untreated or Treated with IFN

A variety of normal and transformed cell lines were either untreated orpre-treated with 100 units of IFN-α, infected at an MOI of 0.1 pfu/mlwith WT Indiana VSV and incubated for 18 hours at 37° C. Culture mediafrom each sample was titred for VSV production. Results are shown inTable 3.

This example demonstrates that viral production is ten to ten thousandtimes more efficient in a range of tumour cell types as compared tonormal primary tissues. In the presence of interferon alpha, virusproduction in normal primary cells is almost completely blocked while intumour cells interferon has little or no effect on VSV replication.TABLE 3 Viral Titre (pfu/ml) Cell Line Untreated IFN-α OSF7 (primarynormal human fibroblast) 1 × 10⁶  <10 OSF12 (primary normal humanfibroblast) 2 × 10⁵  <10 OSF16 (primary normal human fibroblast) 1 × 10⁵ <10 PrEC (primary normal human prostate epithelium) 8 × 10⁶  <10 HOSE(primary normal human ovarian surface 1 × 10⁷ <1000 epithelium) A2780(human ovarian carcinoma) 2 × 10⁸ 1 × 10⁷ OVCA 420 (human ovariancarcinoma) 1 × 10⁸ 3 × 10⁶ C13 (human ovarian carcinoma) 1 × 10⁸ 1 × 10⁵LC80 (human lung carcinoma) 2 × 10⁹ 6 × 10⁷ SK-MEL3 (human melanoma) 1 ×10⁹ 1 × 10⁹ LNCAP (human prostate carcinoma) 4 × 10⁹ 5 × 10⁹ HCT116(human colon carcinoma) 1 × 10⁹ 2 × 10⁹ 293T (HEK cells transformed withT antigen 1 × 10⁸ 8 × 10⁷ and Ad virus E1A)

EXAMPLE 12 LD₅₀ for WT and Mutant VSV Delivered Intranasally toPKR^(+/−) (129×Balb/c) Mice

8-10 week old female mice were anaesthetised and infected intranasallywith virus diluted in 50 μl of phosphate buffered saline (PBS) into thenares of each animal (PKR^(+/−); 129×Balb/c strain). Lethal dose 50values were calculated using the Korler-Spearman method. Results areshown in Table 4.

This example demonstrates that mutants I, II and III in particular, areattenuated as compared to the wild type Indiana strain of virus whentested for toxicity in 129×Balb/c mice. TABLE 4 Virus Intranasal LD₅₀(pfu) WT Indiana  1 × 10⁴ Mutant I  1 × 10¹⁰ Mutant II >1 × 10¹⁰ MutantIII  3 × 10⁸ Mutant IV <1 × 10⁵

EXAMPLE 13 PKR^(−/−) Mice are Exquisitely Sensitive to VSV Compared toVarious PKR^(+/+) Mouse Strains

PKR^(−/−) and PKR^(+/+) mice were infected intranasally at various dosesand their survival monitored over time. All PKR^(−/−) mice succumbed tothe infection between days 2 and 5 depending on the dose, while controlmice remained alive beyond this point. Results are shown in Table 5.

This example demonstrates the importance of the PKR gene product in theresistance of mice to VSV infection. TABLE 5 Genetic Background IN Dose(pfu) Survival at day 5 PKR^(+/+) Balb/c 5 × 10⁴ 5/5 CD-1 5 × 10⁴ 5/5Balb/c × 129 5 × 10⁴ 5/5 PKR^(−/31) Balb/c × 129 5 × 10⁴ 0/5 5 × 10³ 0/45 × 10² 0/3 5 × 10¹ 0/3

EXAMPLE 14 AML3 Cells Die by Apopotosis Following Infection with VSV

OCI/AML3 (acute myelogenous leukemia) cells were infected with VSV at anMOI of 3.0 pfu/cell. Fourteen and twenty hours post-infection unfixedsamples were analyzed. Apoptotic cells with phosphatidylserine membranetranslocation were detected by flow cytometry usingAnnexin-V-Biotin-X/NeutrAvidin-PE red fluorescent protein (MolecularProbes). Mitochondrial membrane depolarization in early apoptotic cellswere analyzed by flow cytometry using JC-1 potential-sensitive dye(Molecular Probes). JC-1 is accumulated by polarized mitochondriashifting fluorescence emission from green to red spectra. Non-viableAML3 cells were identified using Ethidium (EthD-1) homodimer-1 redfluorescent vital dye (Molecular probes). Assays were performedfollowing the manufacturers specifications. Results are shown in Table6.

This example demonstrates that VSV kills AML cells at least in partthrough a virally induced apoptotic pathway. TABLE 6 MOI 0.0 MOI 3.0 NetPercent Percent Positive Tests Positive Positive (Dead) 14 hrs EthD-16.6 32.3 25.7 p.i. Annexin V 14.3 52.7 38.4 JC-1 7.5 21.4 13.4 20 hrsEthD-1 positive 6.4 58.5 52.1 p.i. Annexin-V 10.8 79.6 68.8 positiveJC-1 3.9 43.2 39.3

EXAMPLE 15 Mutant VSV Strains Infect and Kill AML Cells

OCI/AML3 (acute myelogenous leukemia) cells were infected at an MOI of1.0 and incubated for 23 hours. Unfixed cells were stained with Eth-D1(ethidium dimer, Molecular Probes) to detect non-viable cells followingmanufacturers specifications. Number of stained cells per 10,000 countedused to calculate percent dead. Results are shown in Table 7.

This example demonstrates that the mutant VSV strains used are aseffective as the wild type Indiana strain in killing AML cells. TABLE 7WT Mock IND Mut I Mut II Mut III Mut IV Mut V Percent Dead 30.0 64.760.7 86.7 72.1 74.4 82.8

EXAMPLE 16 VSV and VSV Infected Cells Exhibit Antitumor Activity AgainstHuman Melanoma Xenografts in Nude Mice

SK-MEL 3 (melanoma) derived tumours were developed in 8-10 week oldfemale Balb/c athymic mice. On day 0, tumours were either left untreatedor were infected with 10⁸ pfu WT Indiana VSV in culture media or 2.5×10⁶WT Indiana VSV infected SK-MEL 3 cells (VSV producing cells).Statistical differences were calculated between treated and untreatedgroups at each data point with the following confidence values (b:p<0.01; c: p<0.001; d: p=0.007). Results are shown in FIG. 7. On day 3,only tumours treated with VSV producing cells were significantly smallerthan untreated tumours (a: p<0.001). No statistically significantdifferences in tumour volumes between groups were apparent from day 0 today 2. Data points represent means+/−SEM from multiple tumours(untreated n=8; VSV producing cells n=8; VSV alone n=4).

This example demonstrates that a single injection of VSV, directly intosolid tumours profoundly affects tumour growth resulting in partial tocomplete regression. The use of infected tumour cells as a vehicle todeliver virus is also efficacious.

EXAMPLE 17 PKR^(−/−) Mice are Acutely Sensitive to Intranasal VSVInfection and Demonstrate a Deficiency in IFN Mediated Resistance

(A & B) PKR^(−/−) and control mice (Balb/c×129) were infectedintranasally with 5×10⁴ pfu of VSV and monitored for morbidity andsurvival over the course of 14 days, after which remaining animals weredeemed to have survived the infection. Results are shown in FIGS. 8A and8B. PKR^(−/−) mice showed a severe decrease in survival compared tocontrol mice (WT), succumbing by day 3 or 4, while all control micesurvived the infection. IFN-α/β, pretreatment (18 h prior to infection)with either 2×10⁴ IU (FIG. 8A) or 2×10⁵ IU (FIG. 8B) had no protectiveeffect in PKR^(−/−) animals.

This example demonstrates that a single defect in the interferon pathway(absence of PKR gene product) is sufficient to render mice unable toresist VSV infections. This defect cannot be rescued by interferon.

EXAMPLE 18 Interferon Can Protect Xenograft Bearing Nude Mice During VSVTreatment

SK-MEL 3 melanoma cells were injected intradermally into CD-1 athymicnude mice. On day 0, tumours were injected with either live WT IndianaVSV (1×10⁹ pfu) or an equivalent amount of UV inactivated VSV, andmeasured daily. Results are shown in FIG. 9. Interferon was administeredto a subset of animals (VSV IFN) at the times indicated (black arrows).(UV-VSV n=4; VSV IFN n=6; VSV n=6). In these experiments a singleintratumoural injection of VSV is tumour-inhibiting in all cases. Alltumours had at least a partial regression and in three of twelve micetreated a complete tumour regression. Tumours receiving UV inactivatedvirus continued to grow unabated until these animals were sacrificed atday 11. Nude mice not receiving interferon and injected with live virusbegan to die at day 10 and only two of six remained viable by day 15. Incontrast, all interferon treated, infected, nude mice were protectedfrom VSV toxicity and remained symptom free for more than 45 days.

This example demonstrates that a single intratumoural injection of liveVSV is efficacious against tumours. Furthermore infected, tumourbearing, nude mice can be rescued from VSV toxicity by interferoninjection.

EXAMPLE 19 VSV Infects and Kills Leukemia and Myeloma Cells

The indicated cell lines were infected with VSV Indiana HR strain at amultiplicity of infection of one plaque forming unit per cell. At 24, 48and 72 hours post infection (p.i.) samples were taken from the infectedcultures and stained directly with propidium iodide followingmanufacturers instructions (Molecular Probes). Samples were thenanalysed by flow cytometry using the FACSsort WinMDI Version 2.7program. In Table 8 the percentage of cells dead for each leukemic celltype is shown for the indicated times post infection.

This example shows that VSV is able to infect and kill a diverse set ofleukemia types. The K-562 cell is isolated from a chronic myelogenousleukemia (CML) patient while MOLT-4 is a T cell leukemia and SR and H929are myelomas. TABLE 8 Cell Line 24 hr. p.i. 48 hr. p.i. 72 hr. p.i.K-562 (CML) 15.38% 52.36% N/D MOLT-4 (T cell 53.94% 48.80% N/D Leukemia)SR (Myeloma) 32.10% 46.38% N/D H929(Myeloma) 10.73% 17.35% 64.41%

EXAMPLE 20 Vesicular Stomatitis Virus (VSV) Strains Including Wild TypeIndiana and Five Attenuated VSV Mutants Demonstrate SelectiveCytotoxicity Toward Human Prostate Carcinoma Cells Compared to NormalHuman Fibroblasts

Vesicular stomatitis virus strains including wild type Indiana andattenuated mutant strains I (TR1026), II (TR1026R), III (TP3), IV (TP6)and V (G31) were obtained from Dr. Lauren Poliquin, University of Quebecat Montreal. Each of these virus strains was plaque purified five timesprior to use in this experiment.

Human prostate carcinoma cells (LNCAP) and normal human cells (OSF 7forearm fibroblast) were grown in 96-well tissue culture plates to adensity of approximately 5×10⁴ cells per well. Virus was added in10-fold dilutions ranging from 5×10⁵ pfu to 5 pfu. Control wells with novirus were included on each plate. The plates were incubated for 48hours at 37° C. in 5% CO2. Cytotoxicity was quantified using acolorimetric MTS((3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxyphenyl]-2-[4-sulfophenyl]-2H-tetrazolium,inner salt) assay (CellTiter 96 Aqueous, catalog #G1112, PromegaCorporation, Madison Wis. 53711-5399), monitored at 490 nm, that detectsmitochondrial enzyme activity. The amount of cell killing in the virustreated wells was determined by the loss in viability in the virustreated wells relative to the untreated wells. The data was plottedgraphically as pfu/cell vs. percentage cell killing relative to control.The TC50 for these cells was calculated as the amount of virus inpfu/cell causing a 50% reduction in the amount of viable cells. LowerTC50 values reflect increased sensitivity of the cells to the lyticeffects of the virus. The in vitro therapeutic index for each VSV strainwas calculated as the ratio of TC50 for the OSF7 cells compared to theTC50 for the LNCAP cells.

The results are shown in Table 9. Wild type VSV-Indiana and each of thefive mutants demonstrated a high degree of cytotoxicity toward the humanprostate carcinoma cells as reflected in the low TC50 values, all lessthan 0.01 pfu/cell. The normal human fibroblasts cells were one to morethan 3 orders of magnitude more resistant to the cytotoxic effects ofall six VSV strains. All five mutants had less toxicity on the normalOSF7 fibroblasts cells and had a higher in vitro therapeutic index thanthe wild-type Indiana VSV. TABLE 9 Cytotoxicity Assay Results for VSVStrains (Wild type Indiana and Mutants I through V) Against ProstateCarcinoma Cells and Normal Fibroblasts. Mutant Mutant Mutant MutantMutant WT I II III IV V Indiana LNCAP Prostate 0.0064 0.0048 0.00140.0006 0.0012 0.0017 Carcinoma TC50 (pfu/cell) OSF7 NormalFibroblasts >42 22 4.3 0.031 9.8 0.022 TC50 (pfu/cell) TherapeuticIndex >6562 4583 3071 52 8167 13 (TC50 OSF7/ TC50 LNCAPP)

EXAMPLE 21 Virus Production from Tumour Cells and Normal Cells Infectedwith Wild Type Indiana and Various Mutant VSV Strains

HCT 116 colon carcinoma cells and OSF 7 forearm fibroblasts were grownto confluence in 35 mm tissue culture dishes. Media was removed andvirus was added in a volume of 30 μl with a multiplicity of infection of0.1 pfu/cell for the HCT 116 cells and 1.5 pfu/cell for the OSF 7 cells.After a 1 hour incubation period at 37° C., 5% CO2, 1 ml of tissueculture media was added to the dishes. Results are shown in FIGS. 10Aand B.

At the indicated time points, 10 μl samples of media were removed fromthe dishes. The virus titre of these samples was determined by a plaqueassay.

This example demonstrates the rapid replication kinetics of wild typeand mutant VSV strains in HCT 116 colon carcinoma cells. All four mutantVSV strains had more rapid growth in HCT116 tumor cells than the wildtype VSV. Note that in the normal OSF-7 cell cultures a ten fold higherinput of virus is required to attain similar replication kinetics.

EXAMPLE 22 Malignant Cells are Rapidly Killed Following VSV (WT Indiana)Infection and are Not Protected by IFN-α

Monolayers of normal primary human fibroblasts (AG 1522) and severaltumour cell lines were either untreated or pretreated with IFN-α (100units) and then infected with VSV at an MOI of 0.1 pfu/ml. At 12 hoursincrements the infections were terminated by cell fixation and stainingto determine the kinetics of cell killing. Control (CNTL) monolayerswere left to grow, uninfected, over the course of the experiment andtherefore stain more intensely. Results are shown in FIG. 11. LNCAP is ahuman prostate carcinoma; A2780 is an human ovarian epithelialcarcinoma, and Sk MEL3 is a human melanoma.

This example demonstrates the rapid kinetics of tumour cell killing byVSV Indiana even in the presence of interferon alpha. While normal cellsare also killed by VSV, the kinetics are slower and normal cells can becompletely protected by interferon alpha.

EXAMPLE 23 VSV Induced Cytopathic Effect Visible in Human Melanoma Cellsbut Not in Primary Human Cells with or without IFN-α

Gelatin-coated coverslips with normal human cells and SK-MEL3 cellsuntreated or pretreated with IFN-α (100 U/ml) were infected with WTIndiana VSV at an MOI of 0.1 pfu/ml. Results are shown in FIG. 12. Thehuman melanoma cells (SK-MEL3) displayed cpe at 12 hours post-infectioneven in the presence of interferon. At 24 hours post-infection thesemalignant cells had died and lifted from the coverslip. Human primarycells including foreskin fibroblasts (AG1522), ovarian surfaceepithelial cells (HOSE) and prostate epithelial cells (PrEC) did notshow CPE (cytopathic effect) until 36 hours in the absence of interferonand were completely protected in the presence of interferon beyond 72hours post-infection.

This example demonstrates that VSV Indiana is able to rapidly destroymelanoma cells even in the presence of interferon alpha whereas normalfibroblasts and epithelial cells are slower to be killed and can becompletely protected by interferon alpha.

EXAMPLE 24 VSV Selectively Kills Transformed Cells Co-cultured withNormal Fibroblasts

Equal numbers of 293T cells (human embryo kidney cells transformed withadenovirus E1A and Large T antigen) and normal human foreskinfibroblasts were plated on gelatin-coated coverslips and infected (WTIndiana VSV) at an MOI of 0.1 both in the presence and absence ofinterferon. Cells were fixed at 12 (not shown), 24 and 36 hourspost-infection. Fixed cells were stained with an anti-TAg antibody andDAPI. The red staining 293T cells were quickly killed as early as 12hours post-infection, regardless of interferon treatment, with those fewremaining cells displaying condensed or fragmented nuclei. The normalfibroblasts displayed altered nuclei by 36 hours post-infection in theabsence of interferon but were protected from the virus in the presenceof interferon beyond this time point.

This example demonstrates that in a mixed culture of normal and tumourcells, VSV Indiana preferentially replicates and kills tumour cells.Normal cells in the infected co-cultures are slower to die and can becompletely rescued by interferon treatment.

EXAMPLE 25 Efficacy of a Single Intravenous Dose of Mutant VSV inTreating Human Melanoma Xenografts in Nude Mice

SK-Mel3 human melanoma xenografts were established in 5-6 week old CD-1athymic mice. On day 0, tumours were either left untreated or weretreated intravenously with 5×10⁹ pfu of mutant VSV as indicated. Resultsare shown in FIG. 13.

This example demonstrates that mutants II and III are able to inhibittumour growth following a single intravenous injection. Thus virus neednot be administered at the tumour site to be effective in inhibitingtumour growth. Furthermore, the mutants while being attenuated forgrowth in normal mouse tissues, are still able to target tumour cells invivo.

EXAMPLE 26 Selective Killing of AML Cells Co-cultured with Normal BoneMarrow

The growth factor independent cell line OCI/AML3 was mixed 1:9 withnormal bone marrow and infected for 24 hours with WT Indiana VSV.Various dilutions of cells were then plated in methylcellulose plus andminus growth factors and colony counts were performed 14 days later.Table 10 shows data for dishes receiving 10⁴ cells. The asterisk (*)signifies that no leukemic colonies were detected on the growth factorminus dishes even when 10⁵ cells were plated per dish.

This example demonstrates the rapid and selective killing of leukemiacells in the presence of normal bone stem cells. Furthermore itdemonstrates that bone marrow is not a dose-limiting target of VSVoncolytic therapy as it is with most other conventional cancertherapies. TABLE 10 Multiplicity of Infection Colony Type 0.0 1.0 5.0Leukemic 172  0*  0* Neutrophil 12 7 5 Mixed 6 3 4 Monocyte 10 7 5

EXAMPLE 27 VSV Sequences

The genome of VSV contains genes that encode viral proteins N, P, M, Gand L. The cDNA sequences of the open reading frames (ORF) for theseproteins from wild type heat resistant VSV (HR) and three mutant VSVswere determined (based on sequencing five times each) and compared withthe sequences of GenBank Accession No. NC 001560 (derived from Coloradoand San Juan strains of VSV). The mutants are M2 (TR1026R), M3 (TP3) andM4 (TP6). The nucleic acid sequences are shown in FIGS. 14, 16, 18, 20and 22. The corresponding deduced amino acid sequences are shown inFIGS. 15, 17, 19, 21 and 23, respectively. Differences are indicated byhighlighted letters. Dotted lines represent incomplete sequencing.

Some of the differences between the amino acid sequences are shown inTable 11 using notation based on column heading (i.e. for column heading“Differences Between GenBank and HR” notation K155R means that the aminoacid at position 155 is K in GenBank and R in HR). In those cases whereHR sequence is not yet available comparisons can only be made betweenGenBank and a particular mutant. M3* denotes a difference between mutant3 and HR but in this case the amino acid matches the GenBank deposit atthat position (i.e. mutant 3 and the Genbank sequence agree at thatposition while HR is different).

This data demonstrates the many differences in sequence between the HRstrain and the GenBank deposit (which is primarily derived from the SanJuan strain). It also demostrates some of the differences between themutants and the HR strain from which they were derived. These geneticdifferences correlate with phenotypic differences. TABLE 11 DifferencesDifferences Between Differences Between GenBank Differences BetweenBetween HR Genbank and and Gene GenBank and HR and Mutants Mutant #2Mutant #4 N D10A, K155R, N353S A10D (M3*) None (M4) P K50R, A76V, Q77P,None (M2, E99D M3 and M4) P110Q, S126L, S140L Y151H, M168I, E170K D237NM S32N, Y54H, N57H, M51R (M3) T133A, I171V, I226V None (M4) G H24Y,I57L, Q96H, Q26R, A331V V141A, Y172D, R242H, G132D S431A H242R, S438T,L453F (all M3) H487Y E254G (M4) L T367A, T689S, None (M4) I202L, T2026IK296R R2075K

All citations are herein incorporated by reference.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

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1. A method of reducing the viability of a tumor cell, comprisingadministering to the tumor cell an attenuated strain of vesicularstomatitis virus, wherein said tumor cell is a carcinoma.
 2. The methodof claim 1, wherein the carcinoma is a lung carcinoma.
 3. The method ofclaim 1, wherein the tumor cell is PKR−/−; STAT1−/−; or both PKR−/− andSTAT1−/−.
 4. The method of claim 1, wherein the virus is unable toinactivate PKR activity within the tumor cell.
 5. The method of claim 1,wherein the virus is vesicular stomatitis virus strain M1.
 6. The methodof claim 1, wherein the virus is vesicular stomatitis virus strain M2.7. The method of claim 1, wherein the virus is vesicular stomatitisvirus strain M3.
 8. The method of claim 1, wherein the virus isvesicular stomatitis virus strain M4.
 9. The method of claim 1, whereinthe virus is vesicular stomatitis virus strain M5.
 10. The method ofclaim 1, wherein the tumor cell is in a mammalian subject and the virusis administered to the tumor cell by intravenous, intranasal,intraperitoneal or intratumoral administration to the subject.
 11. Themethod of claim 10, wherein the mammalian subject is a human or anon-human mammal.
 12. The method of claim 10, wherein the virus iscontained in cell line infected with the virus and the administrationcomprises administering the virus-infected cell line to the subject by aroute selected from intratumorally, intravenously or intraperitoneally.13. A method of reducing the viability of a tumor cell within apopulation of tumor cells and non-tumor cells comprising administeringan attenuated strain of vesicular stomatitis virus to the population ofcells, wherein tumor cells are carcinoma cells and the virus is able toselectively infect and kill the tumor cell.
 14. The method of claim 13,wherein the virus is unable to inactivate PKR activity in the tumorcell.
 15. The method of claim 14, further comprising treating thepopulation of cells with interferon prior to administering the virus.